Limiting yeast-produced trehalose in fermentation

ABSTRACT

The present disclosure relates to recombinant yeast host cells having (i) a first genetic modification for reducing the production of one or more native enzymes that function to produce glycerol or regulating glycerol synthesis and/or allowing the production of an heterologous glucoamylase and (ii) a second genetic modification for reducing the production of one or more native enzymes that function to produce trehalose or regulating trehalose synthesis and/or allowing the expression of an heterologous trehalase. The recombinant yeast host cells can be used to limit the production of (yeast-produced) trehalose (particularly extracellular trehalose) during fermentation and, in some embodiments, can increase the production of a fermentation product (such as, for example, ethanol).

CROSS-REFERENCE TO RELATED APPLICATIONS AND DOCUMENTS

This application claims priority from U.S. provisional patentapplication 62/251,885 filed on Nov. 6, 2015. This application is filedconcurrently with a sequence listing in an electronic format. Thecontent of the priority application and the sequence listing is herewithincorporated in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 580127_418C3_SEQUENCE_LISTING.txt. The text fileis 145 KB, was created on Feb. 8, 2021, and is being submittedelectronically via EFS-Web.

TECHNOLOGICAL FIELD

This disclosure relates to the use of recombinant yeast host cells forlimiting the production and/or secretion of trehalose duringfermentation, such as, for example, an alcoholic fermentation. The useof the recombinant yeast host cells can increase the production of oneor more fermentation product, such as, for example, ethanol.

BACKGROUND

Saccharomyces cerevisiae is the primary biocatalyst used in thecommercial production of fuel ethanol. This organism is proficient infermenting glucose to ethanol, often to concentrations greater than 20%w/v. However, S. cerevisiae lacks the ability to hydrolyzepolysaccharides and therefore may require the exogenous addition ofenzymes to convert complex sugars to glucose. Several S. cerevisiaestrains have been genetically engineered to express alpha(α)-amylaseand/or glucoamylase (see for example, WO 2011/153516 and WO 2012/138942)and the use of such strains increases the overall yield whilerepresenting a substantial cost savings.

One potential for yield improvements is targeting the breakdown ofresidual fermentable sugars. For example, a typical corn ethanolfermentation will have approximately 4 g/L of residual disaccharidesugars (also referred to as degree of polymerization 2 or DP2),comprised of maltose, isomaltose and the majority being trehalose(Giffen 2012). These disaccharides represent a potential of anadditional 4 g/L ethanol.

Trehalose is a non-reducing disaccharide composed of two glucosemolecules linked at the 1-carbon, forming an α-α bond. In yeasts,trehalose can act as carbohydrate storage, but more importantly, it hasbeen well characterized to act as a stress protectant againstdesiccation, high temperatures, ethanol toxicity, and acidic conditionsby stabilizing biological membranes and native proteins (Elbein et al.2003; Singer and Lindquist 1998). Intracellular trehalose iswell-regulated in yeast based on an equilibrium between synthesis anddegradation. It is obtained by catalyzing the combination of auridine-diphosphate-glucose moiety to a glucose-6-phosphate to formtrehalose-6-phosphate (see FIG. 1). The phosphate group is then removedto form trehalose. The primary pathway is facilitated by a proteincomplex encoded by four genes: the trehalose-6-phosphate synthase (TPS1encoded by the tps1 gene), trehalose-6-phosphate phosphatase (TPS2encoded by the tps2 gene) and two regulatory proteins, TPS3 and TSL1.Trehalose can be catabolized into two glucose molecules by either thecytoplasmic trehalase enzyme, NTH1, or the tethered, extracellulartrehalase, ATH1. Under certain conditions, trehalose can also beexcreted from the yeast.

The trehalose biosynthetic pathway has also been proposed to be aprimary regulator of glycolysis by creating a futile cycle. As glucoseis phosphorylated by hexokinase (HXK), the intracellular free organicphosphate levels are quickly depleted which is required for downstreamprocesses and other metabolic processes (see FIG. 1). Conversion ofglucose-6-phosphate into trehalose not only removes the sugar fromglycolysis, creating a buffer, but the pathway also regeneratesinorganic phosphate. Another primary control of glycolysis is theinhibition of HXK2 by trehalose-6-phosphate, thereby further slowing theglycolysis flux.

Numerous manipulations of the trehalose pathway in S. cerevisiae havebeen reported. Attempts at trehalose manipulations as a means oftargeting ethanol yield increases have primarily focused onover-expression of the pathway, particularly with TPS1/TPS2 (Cao et al.2014; Guo et al. 2011; An et al. 2011). Ge et al. (2013) successfullyimproved ethanol titers on pure glucose with the over-expression of theTSL1 component, which has also been implicated in glucose signaling.Deletion of the biosynthetic pathway has proved more challenging. Asreviewed by Thevelein and Hohmann (1995), attempts to remove the TPS1function have led to the decreased ability to grow on readilyfermentable carbon sources due to the aforementioned control ofglycolysis. Functional analysis of the TPS complex has been extensivelystudied using knockout approaches (Bell et al. 1998), but none havetargeted deletion of the key biosynthetic genes as a means of improvingethanol yields nor have they targeted relevant fuel ethanol processes.

It would be highly desirable to be provided with methods of usingrecombinant yeast host cells which are capable of modulating theproduction and/or the excretion of trehalose and/or trehalose breakdownwhile being capable of fermenting a medium and producing a fermentationproduct.

BRIEF SUMMARY

The present disclosure relates to the use of recombinant yeasts capableof limiting the accumulation of trehalose during fermentation in orderto increase the production of another fermentation product duringfermentation. The recombinant yeast host cells comprises at least twogenetic modifications. The first genetic modification allows forreducing the production of one or more native enzymes that function toproduce glycerol or regulating glycerol synthesis and/or allowing theproduction of an heterologous glucoamylase. The second geneticmodification allows for reducing the production of one or more nativeenzymes that function to produce or regulating trehalose synthesisand/or allowing the expression of an heterologous trehalase.

In a first aspect, the present disclosure provides a recombinant yeasthost cell comprising: (i) a first genetic modification for reducing theproduction of one or more native enzymes that function to produceglycerol or regulating glycerol synthesis and/or allowing the productionof an heterologous glucoamylase; and (ii) a second genetic modificationfor reducing the production of one or more native enzymes that functionto produce trehalose or regulating trehalose synthesis and/or allowingthe expression of an heterologous trehalase. In an embodiment, therecombinant yeast host cell has the second genetic modification allowingthe expression of the heterologous trehalase. In an embodiment, therecombinant yeast host cell has the first genetic modification forreducing the production of the one or more native enzymes that functionto produce glycerol and the second genetic modification for reducing theproduction of the one or more native enzymes that function to producetrehalose or regulating trehalose synthesis. In another embodiment, therecombinant yeast host cell has the first modification for reducing theproduction of the one or more native enzymes that function to produceglycerol or regulating glycerol synthesis and the second modificationallowing the production of the heterologous trehalase. In yet anotherembodiment, the recombinant yeast host cell has the first geneticmodification allowing the production of the heterologous glucoamylaseand the second genetic modification for reducing the production of theone or more native enzymes that function to produce trehalose orregulating trehalose synthesis. In still another embodiment, therecombinant yeast host cell has the first genetic modification allowingthe production of the heterologous glucoamylase and the second geneticmodification allowing the production of the heterologous trehalase. In afurther embodiment, the recombinant yeast host cell comprises a further(third) genetic modification for reducing the production of the one ormore native enzymes that function to catabolize formate. In suchembodiment, the recombinant yeast host cell can, for example, lack theability to produce a FDH1 polypeptide and/or a FDH2 polypeptide. In anembodiment, the recombinant yeast host cell comprises a further (fourth)genetic modification to reduce the production of the one or more nativeenzymes that function to produce glycerol (e.g., decreases or inhibitsin the expression of the a GPD1 polypeptide and/or a GPD2 polypeptide)or regulating glycerol synthesis (e.g., decreases or inhibits theexpression of a FPS1 polypeptide and/or increases the expression of aSTL1 polypeptide). In another embodiment, the recombinant yeast hostcell comprises a further (fifth) genetic modification allowing theexpression of heterologous glucoamylase. In an embodiment, theheterologous glucoamylase is from the genus Saccharomycopsis sp., suchas, for example, from the species Saccharomycopsis fibuligera. In yet afurther embodiment, the heterologous glucoamylase has or consists of theamino acid sequence of SEQ ID NO: 3, is a variant of the amino acidsequence of SEQ ID NO: 3 or is a fragment of the amino acid sequence ofSEQ ID NO: 3. In another embodiment, the second genetic mutation causesa reduction in the expression level or prevents the expression of theone or more native enzymes that function to produce trehalose orregulating trehalose synthesis, such as, for example, the TPS2polypeptide or a polypeptide encoded by a tps2 ortholog. In a furtherembodiment, the heterologous trehalase is an acid trehalase. In afurther embodiment, the acid trehalase is from the genus Aspergillussp., for example, from the species Aspergillus fumigatus or the speciesAspergillus nidulans. In an embodiment, the acid trehalase has the aminoacid sequence of SEQ ID NO: 1, is a variant of the amino acid sequenceof SEQ ID NO: 1 or is a fragment of the amino acid sequence of SEQ IDNO: 1. In yet another embodiment, the acid trehalase has the amino acidsequence of SEQ ID NO: 2, is a variant of the amino acid sequence of SEQID NO: 2 or is a fragment of the amino acid sequence of SEQ ID NO: 2. Instill another embodiment, the recombinant yeast host cell is from thegenus Saccharomyces sp., such as, for example, from the speciesSaccharomyces cerevisiae.

In a second aspect, the present disclosure provides a method of limitingthe accumulation of extracellular trehalose during a fermentation.Broadly, the method comprises fermenting a medium with at least onerecombinant yeast host cell described herein. In an embodiment, themethod can also include adding an heterologous trehalase to the medium.

In a third aspect, the present disclosure provides a method ofincreasing the production of a fermentation product during afermentation, said method comprising fermenting a medium with at leastone recombinant yeast host cell described herein. In an embodiment, themethod can also include adding an heterologous acid trehalase to themedium. In a further embodiment, the fermentation product is ethanol. Inyet another embodiment, the medium comprises starch which can beoptionally be provided in a gelatinized or a raw form. In yet anotherembodiment, the medium can be derived from corn. In another embodiment,the medium comprises maltodextrin.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, referencewill now be made to the accompanying drawings, showing by way ofillustration, a preferred embodiment thereof, and in which:

FIG. 1 illustrates the trehalose synthesis pathway. Abbreviations:HXK=hexokinase; GLK=glucokinase; PGM=phosphoglucomutase;UGP1=UDP-glucose pyrophosphorylase; GSY=glycogen synthase; GPH=Glycogenphosphorylase; TPS1=trehalose-6-phosphate synthase;TPS3=trehalose-6-phosphate synthase; TSL1=trehalose synthase long chain;TPS2=trehalose-6-phosphate phosphatase; NTH=neutral trehalase; ATH1=acidtrehalase.

FIG. 2 illustrates the effect of TPS1 (M4652) and TPS2 (M4653) knockoutson ethanol production in 20% corn mash fermentation compared to theconventional parent strain (M2390). Results are shown as ethanolconcentration (in g/L) in function of strains used.

FIG. 3 illustrates the effect of TPS1 (M4652 or ▪) and TPS2 (M4653 or ▴)knockouts on trehalose production in 20% corn mash fermentation comparedto the conventional parent strain (M2390 or ♦). Results are shown astrehalose concentration (in g/L) in function of strains used.

FIG. 4 illustrates the results of a secreted trehalase assay of strainsexpressing various heterologous trehalases. The results are provided asabsorbance at 450 nm in function of strain used. The parental strainM2390 was used as a control. Results are shown were for the followingheterologous trehalase MP241 (SEQ ID NO: 4), MP242 (SEQ ID NO: 5), MP243(SEQ ID NO: 6), MP244 (SEQ ID NO: 1), MP245 (SEQ ID NO: 7), MP246 (SEQID NO: 8), MP841 (SEQ ID NO: 9), MP842 (SEQ ID NO: 10), MP843 (SEQ IDNO: 11), MP844 (SEQ ID NO: 12), MP845 (SEQ ID NO: 13), MP846 (SEQ ID NO:14), MP847 (SEQ ID: 15), MP848 (SEQ ID NO: 2), MP850 (SEQ ID NO: 16),MP851 (SEQ ID NO: 17) and MP853 (SEQ ID NO: 18). MP244 and MP848 wereidentified as the most active when compared to the parental (negativecontrol) strain M2390.

FIG. 5 illustrates the effect of supplementing a fermentation mediumwith a trehalase on ethanol production. A 25.5% corn mash fermentationwas conducted comparing the conventional strain (M2390) to a straingenetically engineered to express a glucoamylase strain (M8841) with andwithout the addition of purified yeast-made trehalase (MP244). M2390received a 100% dose of commercial GA, whereas both M8841 treatmentsreceived a 50% GA dose. The M8841+MP244 fermentations received 100 μg/mlof purified MP244. Results are shown as ethanol concentration (in g/L)in function of experimental conditions used.

FIG. 6 illustrates the effect of supplementing a fermentation mediumwith a trehalase on residual trehalose at the end of a 25.5% corn mashfermentation (described in the legend of FIG. 5). Results are shown astrehalose concentration (in g/L) in function of experimental conditionsused.

FIGS. 7A and 7B illustrate the effects of fermenting a fermentationmedium by a S. cerevisiae strain expressing an heterologous glucoamylaseand an heterologous trehalase on ethanol production (A) and trehaloseconcentration (B). Results are shown as ethanol concentration (A, ing/L) or trehalose concentration (B, in g/L) in function of experimentalconditions used.

FIG. 8A to 8E illustrate the genetic maps of the different cassettesused to generate some of the recombinant yeast strains of the Examples.(A) Map of the MA613 cassette used for making the M4652 strain. KT-MXand NT-MX cassette used to knock-out the tps1 open reading frame. Thecassette contains the positive selection markers kanamycin gene ornourseothricin gene under control of the Ashbya gossypii tef promoterand terminator along with the HSV-thymidine kinase negative selectionmarker under control of the S. cerevisiae hxt2 promoter and act1terminator. (B) Map of the MA614 cassette used for making the M4653strain. KT-MX and NT-MX cassette used to knock-out the tps2 open readingframe. The cassette contains the positive selection markers kanamycingene or nourseothricin gene under control of the Ashbya gossypii tefpromoter and terminator along with the HSV-thymidine kinase negativeselection marker under control of the S. cerevisiae hxt2 promoter andact1 terminator. (C) Map of the MA1920 cassette for making the M11245strain and integrating the MP244 trehalase at the FCY1 locus undercontrol of the native S. cerevisiae tef2 promoter and adh3 terminator.(D) Map of the MAP516 cassette for making the M10957 strain and forintegrating the MP848 trehalase at the FCY1 locus under control of thenative S. cerevisiae tef2 promoter and idp1 terminator. (E) Map of theMAP811 cassette for making the M12121 and M13913 strains and forintegrating MP244 trehalase at the IME1 locus under control of thenative S. cerevisiae tef2 promoter and idp1 terminator.

FIGS. 9A and 9B illustrate the effects of a maltodextrin fermentation bya S. cerevisiae strain M13913 expressing an heterologous glucoamylase,the glycerol reduction pathway as described in WO 2012/138942 and anheterologous trehalase on ethanol (A) and trehalose (B) concentrations.Results are shown as ethanol concentration (A, in g/L) or trehaloseconcentration (B, in g/L) in function of experimental conditions used.

DETAILED DESCRIPTION

The present disclosure relates to the use of recombinant yeast hostcells capable of limiting the production, accumulation or excretion oftrehalose during fermentation. The recombinant yeast host cellcomprising at least two distinct genetic modifications (also referred toas genetic mutations). The genetic modifications are preferably madeusing genetic engineering techniques. Firstly, the recombinant yeasthost cells can be modified to reduce or inhibit the production of one ormore native enzymes that function to produce glycerol or regulatingglycerol synthesis. Alternatively or in combination, the recombinantyeast host cells can be modified to produce an heterologousglucoamylase. Secondly, the recombinant yeast host cells can be modifiedto reduce or inhibit the production of one or more native enzymes thatfunction to produce trehalose or regulating trehalose synthesis.Alternatively or in combination, the recombinant yeast host cells can bemodified to produce an heterologous trehalase. The use of suchrecombinant yeast host cells, in some conditions, limits the level oftrehalose during fermentation to a maximum of 1.0 g/L.

Recombinant Yeast Host Cells

The present disclosure concerns recombinant yeast host cells that havebeen genetically engineered to include at least two geneticmodifications. The genetic modifications can be made in one or bothcopies of the targeted gene(s). In the context of the presentdisclosure, when recombinant yeast cell is qualified as being“genetically engineered”, it is understood to mean that it has beenmanipulated to either add at least one or more heterologous or exogenousnucleic acid residue and/or removed at least one endogenous (or native)nucleic acid residue. The genetic manipulations did not occur in natureand are the results of in vitro manipulations of the yeast.

In the context of the present disclosure, the recombinant host cell is ayeast. Suitable yeast host cells can be, for example, from the genusSaccharomyces, Kluyveromyces, Arxula, Debaryomyces, Candida, Pichia,Phaffia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces orYarrowia. Suitable yeast species can include, for example, S.cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S.diastaticus, K. lactis, K. marxianus or K. fragilis. In someembodiments, the yeast is selected from the group consisting ofSaccharomyces cerevisiae, Schizzosaccharomyces pombe, Candida albicans,Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenulapolymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans,Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomycespombe and Schwanniomyces occidentalis. In one particular embodiment, theyeast is Saccharomyces cerevisiae. In some embodiment, the host cell canbe an oleaginous yeast cell. For example, the oleaginous yeast host cellcan be from the genus Blakeslea, Candida, Cryptococcus, Cunninghamella,Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum,Rhodotorula, Trichosporon or Yarrowia. In some alternative embodiment,the host cell can be an oleaginous microalgae host cell (e.g., forexample, from the genus Thraustochytrium or Schizochytrium). In anembodiment, the recombinant yeast host cell is from the genusSaccharomyces and, in some embodiments, from the species Saccharomycescerevisiae.

The first modification of the recombinant yeast host cell can be agenetic modification leading to the reduction in the production, and inan embodiment to the inhibition in the production, of one or more nativeenzymes that function to produce glycerol or regulating glycerolsynthesis. As used in the context of the present disclosure, theexpression “reducing the production of one or more native enzymes thatfunction to produce glycerol or regulating glycerol synthesis” refers toa genetic modification which limits or impedes the expression of genesassociated with one or more native polypeptides (in some embodimentsenzymes) that function to produce glycerol or regulating glycerolsynthesis, when compared to a corresponding yeast strain which does notbear the first genetic modification. In some instances, the firstgenetic modification reduces but still allows the production of one ormore native polypeptides that function to produce glycerol or regulatingglycerol synthesis. In other instances, the first genetic modificationinhibits the production of one or more native enzymes that function toproduce glycerol or regulating glycerol synthesis. In some embodiments,the recombinant yeast host cells bear a plurality of first geneticmodifications, wherein at least one reduces the production of one ormore native polypeptides and at least another inhibits the production ofone or more native polypeptides. As used in the context of the presentdisclosure, the expression “native polypeptides that function to produceglycerol or regulating glycerol synthesis” refers to polypeptides whichare endogenously found in the recombinant yeast host cell. Nativeenzymes that function to produce glycerol include, but are not limitedto, the GPD1 and the GPD2 polypeptide (also referred to as GPD1 and GPD2respectively). Native enzymes that function to regulating glycerolsynthesis include, but are not limited to, the FPS1 polypeptide as wellas the STL1 polypeptide. The FPS1 polypeptide is a glycerol exporter andthe STL1 polypeptide functions to import glycerol in the recombinantyeast host cell. By either reducing or inhibiting the expression of theFPS1 polypeptide and/or increasing the expression of the STL1polypeptide, it is possible to control, to some extent, glycerolsynthesis. In an embodiment, the recombinant yeast host cell bears agenetic modification in at least one of the gpd1 gene (encoding the GPD1polypeptide), the gpd2 gene (encoding the GPD2 polypeptide), the fps1gene (encoding the FPS1 polypeptide) or orthologs thereof. In anotherembodiment, the recombinant yeast host cell bears a genetic modificationin at least two of the gpd1 gene (encoding the GPD1 polypeptide), thegpd2 gene (encoding the GPD2 polypeptide), the fps1 gene (encoding theFPS1 polypeptide) or orthologs thereof. In still another embodiment, therecombinant yeast host cell bears a genetic modification in each of thegpd1 gene (encoding the GPD1 polypeptide), the gpd2 gene (encoding theGPD2 polypeptide) and the fps1 gene (encoding the FPS1 polypeptide) ororthologs thereof. Examples of recombinant yeast host cells bearing suchgenetic modification(s) leading to the reduction in the production ofone or more native enzymes that function to produce glycerol orregulating glycerol synthesis are described in WO 2012/138942.Preferably, the recombinant yeast host cell has a genetic modification(such as a genetic deletion or insertion) only in one enzyme thatfunctions to produce glycerol, in the gpd2 gene, which would cause thehost cell to have a knocked-out gpd2 gene. In some embodiments, therecombinant yeast host cell can have a genetic modification in the gpd1gene, the gpd2 gene and the fps1 gene resulting is a recombinant yeasthost cell being knock-out for the gpd1 gene, the gpd2 gene and the fps1gene. In still another embodiment (in combination or alternative to the“first” genetic modification described above), the recombinant yeasthost cell can have a genetic modification in the st1 gene (e.g., aduplication for example) for increasing the expression of the STL1polypeptide.

The first genetic modification can also allow for the production of anheterologous glucoamylase. Many microbes produce an amylase to degradeextracellular starches. In addition to cleaving the last α(1-4)glycosidic linkages at the non-reducing end of amylose and amylopectin,yielding glucose, γ-amylase will cleave α(1-6) glycosidic linkages. Theheterologous glucoamylase can be derived from any organism. In anembodiment, the heterologous protein is derived from a γ-amylase, suchas, for example, the glucoamylase of Saccharomycoces filbuligera (e.g.,encoded by the glu 0111 gene). Examples of recombinant yeast host cellsbearing such first genetic modifications are described in WO2011/153516.

The heterologous glucoamylase can be a variant of a known glucoamylase,for example a variant of the heterologous glucoamylase having the aminoacid sequence of SEQ ID NO: 3. The glucoamylase variants have at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to the glucoamylases described herein. A variant comprises atleast one amino acid difference when compared to the amino acid sequenceof the native glucoamylase. The term “percent identity”, as known in theart, is a relationship between two or more polypeptide sequences or twoor more polynucleotide sequences, as determined by comparing thesequences. The level of identity can be determined conventionally usingknown computer programs. Identity can be readily calculated by knownmethods, including but not limited to those described in: ComputationalMolecular Biology (Lesk, A. M., ed.) Oxford University Press, N Y(1988); Biocomputing: Informatics and Genome Projects (Smith, D. W.,ed.) Academic Press, N Y (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, N J(1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.)Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. andDevereux, J., eds.) Stockton Press, NY (1991). Preferred methods todetermine identity are designed to give the best match between thesequences tested. Methods to determine identity and similarity arecodified in publicly available computer programs. Sequence alignmentsand percent identity calculations may be performed using the Megalignprogram of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Multiple alignments of the sequences disclosed hereinwere performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PEN ALT Y=10). Default parameters for pairwise alignmentsusing the Clustal method were KTUPLB 1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5. The variant heterologous glucoamylases describedherein may be (i) one in which one or more of the amino acid residuesare substituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide forpurification of the polypeptide.

A “variant” of the glucoamylase can be a conservative variant or anallelic variant. As used herein, a conservative variant refers toalterations in the amino acid sequence that do not adversely affect thebiological functions of the glucoamylase. A substitution, insertion ordeletion is said to adversely affect the protein when the alteredsequence prevents or disrupts a biological function associated with theglucoamylase (e.g., the hydrolysis of starch). For example, the overallcharge, structure or hydrophobic-hydrophilic properties of the proteincan be altered without adversely affecting a biological activity.Accordingly, the amino acid sequence can be altered, for example torender the peptide more hydrophobic or hydrophilic, without adverselyaffecting the biological activities of the glucoamylase.

The heterologous glucoamylase can be a fragment of a known glucoamylaseor fragment of a variant of a known glucoamylase (such as, for example,a fragment of the glucoamylase having the amino acid sequence of SEQ IDNO: 3). Glucoamylase “fragments” have at least at least 100, 200, 300,400, 500 or more consecutive amino acids of the glucoamylase. A fragmentcomprises at least one less amino acid residue when compared to theamino acid sequence of the glucoamylase and still possess the enzymaticactivity of the full-length glucoamylase. In some embodiments, fragmentsof the glucoamylases can be employed for producing the correspondingfull-length glucoamylase by peptide synthesis. Therefore, the fragmentscan be employed as intermediates for producing the full-length proteins.

The heterologous nucleic acid molecule encoding the heterologousglucoamylase, variant or fragment can be integrated in the genome of theyeast host cell. The term “integrated” as used herein refers to geneticelements that are placed, through molecular biology techniques, into thegenome of a host cell. For example, genetic elements can be placed intothe chromosomes of the host cell as opposed to in a vector such as aplasmid carried by the host cell. Methods for integrating geneticelements into the genome of a host cell are well known in the art andinclude homologous recombination. The heterologous nucleic acid moleculecan be present in one or more copies in the yeast host cell's genome.Alternatively, the heterologous nucleic acid molecule can beindependently replicating from the yeast's genome. In such embodiment,the nucleic acid molecule can be stable and self-replicating.

In the context of the present disclosure, the recombinant yeast hostcell can include at least two “first” genetic modifications, one inleading to the reduction in the production of one or more native enzymesthat function to produce glycerol or regulating glycerol synthesis andanother one leading to the expression of an heterologous glucoamylase.It is also contemplated that the recombinant yeast host cell can includea single first genetic modification, either for reducing in theproduction of one or more native enzymes that function to produceglycerol or regulating glycerol synthesis or for expressing anheterologous glucoamylase.

The second genetic modification of the recombinant yeast host cell canlead to the reduction in the production (or the prevention ofexpression) of one or more native enzymes that function to producetrehalose or regulating trehalose synthesis. As used in the context ofthe present disclosure, the expression “reducing the production of oneor more native enzymes that function to produce trehalose or regulatingtrehalose synthesis” refers to a genetic modification which limits orimpedes the expression of genes associated with one or more nativepolypeptides (in some embodiments enzymes) that function to producetrehalose or regulating trehalose synthesis, when compared to acorresponding yeast strain which does not bear the second geneticmodification. In some instances, the second genetic modification reducesbut still allows the production of one or more native polypeptides thatfunction to produce trehalose or regulating trehalose synthesis. Inother instances, the second genetic modification inhibits the productionof one or more native enzymes that function to produce trehalose orregulating trehalose synthesis. In some embodiments, the recombinantyeast host cells bear a plurality of second genetic modifications,wherein at least one reduces the production of one or more nativepolypeptides and at least another inhibits the production of one or morenative polypeptides. As used in the context of the present disclosure,the expression “native polypeptides that function to produce trehaloseor regulating trehalose synthesis” refers to polypeptides which areendogenously found in the recombinant yeast host cell. Native enzymesthat function to produce trehalose include, but are not limited to, theTPS1 and TPS2 (both members of the TPS complex). Native enzymes thatfunction to regulating trehalose synthesis include, but are not limitedto polypeptides involved in interacting with the TPS complex such as,for example, TPS3 and TSL1 as well as polypeptides responsible forsynthesizing precursors of the TPS complex such as, for example, NTH1,NTH2, ATH1, HXK1, HXK2, GLK1, PGM1, PGM2, GPH1, UGP1, GSY1 and GSY2. Inan embodiment, the recombinant yeast host cell bears a geneticmodification in at least one, two, three or more of the tps1 gene(encoding the TPS1 polypeptide), the tps2 gene (encoding the TPS2polypeptide), the tps3 gene (encoding the TPS3 polypeptide), the tsl1gene (encoding the TSL1 polypeptide), the nth1 gene (encoding the NTH1polypeptide), the nth2 gene (encoding the NTH2 polypeptide), the ath1gene (encoding the ATH1 polypeptide), the hxk1 gene (encoding the HXK1polypeptide), the hxk2 gene (encoding the HXK2 polypeptide), the g/k1gene (encoding the GLK1 polypeptide), the pgm1 gene (encoding the PGM1polypeptide), the pgm2 gene (encoding the PGM2 polypeptide), the gph1gene (encoding the GPH1 polypeptide), the ugp1 gene (encoding the UGP1polypeptide), the gsy1 gene (encoding the GSY1 polypeptide), the gsy2gene (encoding the GSY2 polypeptide) or orthologs thereof. Preferably,the recombinant yeast host cell has a genetic modification (such as agenetic deletion or insertion) only in one enzyme that functions toproduce glycerol, in the tps2 gene, which would cause the host cell tohave a knocked-out tps2 gene.

In some circumstances, the second genetic modification can be made to(or, in some instances, limited to) the tps2 gene or to the tps2 geneortholog. As such, the recombinant yeast host cell can lack the abilityto produce a biologically active trehalose-6-phosphate phosphatase (TPS2polypeptide). The yeast strain can be genetically engineered to impedeor prevent the expression of the tps2 gene or to allow the expression ofa non-functional TPS2 polypeptide. In an embodiment, the second geneticmodification can be limited to the tps2 gene (or its ortholog), itscorresponding transcript or its corresponding polypeptide and areintended to either reduce the expression of the gene, reduce theexpression and/or stability of the transcript, reduce the expressionand/or stability of the polypeptide or reduce the biological activity ofthe polypeptide. In one embodiment, the open-reading frame of the tps2gene (or its ortholog) is disrupted specifically by the introduction ofan heterologous nucleic acid molecule. In another embodiment, theopen-reading frame of the tps2 gene can be deleted (in part or intotal).

In some instances, the recombinant yeast host cell can have the abilityto produce trehalose-6-phosphate (for example by producing the TPS1polypeptide). In the context of the present disclosure, the expression“capable of producing trehalose-6-phosphate” refers to a yeast strainwhich has the ability of expressing a gene or a combination of genesleading to the production of trehalose-6-phosphate. Thetrehalose-6-phosphate synthase gene (also referred to the tps1 gene) andthe activity of the trehalose-6-phosphate synthase (referred to as theTPS1 polypeptide) are important in the production oftrehalose-6-phosphate. As such, a recombinant yeast strain capable ofproducing trehalose-6-phosphate usually has a tps1 gene and is capableof expressing a functional/biologically active TPS1 polypeptide. As itis known in the art, the TPS1 polypeptide is an enzyme involved in thesynthesis of trehalose-6-phosphate from UDP-glucose.

Still in the context of the present disclosure, the expression “lackingthe ability to produce a biologically active trehalose-6-phosphatephosphatase (TPS2 polypeptide)” refers to a yeast strain which has beengenetically engineered to prevent the expression from the tps2 gene orexpresses a non-functional trehalose-6-phosphate phosphatase (TPS2polypeptide). As known in the art, the tps2 gene encodes an enzyme (TPS2polypeptide) having the ability to recognize trehalose-6-phosphate andcleave the bound between trehalose and the phosphate group. As such, abiologically active or functional TPS2 polypeptide is capable ofrecognizing trehalose-6-phosphate and cleave the bound between trehaloseand the phosphate group. It follows that a biologically inactive ornon-functional TPS2 polypeptide cannot recognize trehalose-6-phosphateand/or cleave the bound between trehalose and the phosphate group.

As indicated above, the recombinant yeast host cell can be geneticallyengineered to impede or prevent the expression of the tps2 gene (or atps2 gene ortholog) by manipulating the endogenous coding sequence ofthe nucleic acid sequence of the tps2 gene (or the tps2 gene ortholog).The tps2 gene (also known as hog2 or pfk3) has been specificallydescribed in Saccharomyces cerevisiae and is associated with the Gene ID851646. In the context of the present disclosure, a “tps2 gene ortholog”is understood to be a gene in a different species that evolved from acommon ancestral gene by speciation. In the context of the presentinvention, a tps2 ortholog retains the same function, e.g. it encodesfor an enzyme capable of dephosphorylating trehalose-6-phosphate.

The TPS2 polypeptide has been specifically described in Saccharomycescerevisiase under GenBank Accession Number CAA98893.1. In the context ofthe present disclosure, the tps2 gene (or its ortholog) can encode aTPS2 polypeptide having one of the following GenBank Accession NumberXP_009255856.1, CEP23739.1, EKJ75382.1, CAA98893.1, P31688.3 GI:1730010,014145.1, NP_010359.1, DAA11920.1, CAB16285., NP_594975.1, CAA86796.1,AAF80562.1, CAA50025.1, CDM32404.1, BA038481.1, AJV20879.1, AJV20163.1,AJV19466.1, AJV18745.1, AJV18033.1, AJV17324.1, AJV16619.1, AJV15908.1,AJV15200.1, AJV14492.1, AJV13824.1, AJV13114.1, AJV12465.1, AJV11777.1,AJV11078.1, AJV10431.1, AJV09728.1, AJV09023.1, AJV08338.1, AJV07644.1,AJV06938.1, AJV06235.1, AJV05531.1, AJV04812.1, AJV04100.1, AJV03395.1,AJV02725.1, AJV02019.1, AJV01306.1, AJV00594.1, AJU99880.1, AJU99185.1,AJU98485.1, AJU97772.1, AJU97074.1, AJU96370.1, AJU95666.1, AJU94967.1,AJU94268.1, AJU93563.1, AJU92846.1, AJU92131.1, AJU91414.1, AJU90697.1,AJU89979.1, AJU89269.1, AJU88578.1, AJU87873.1, AJU87202.1, AJU86553.1,AJU85852.1, AJU85155.1, AJU84444.1, AJU83731.1, AJU83018.1, AJU82439.1,AJU81736.1, AJU81046.1, AJU80346.1, AJU79746.1, AJU79035.1, AJU78323.1,AJU77611.1, AJU76901.1, AJU76191.1, AJU75483.1, AJU74777.1, AJU74061.1,AJU73348.1, AJU72635.1, AJU71925.1, AJU71213.1, AJU70526.1, AJU69816.1,AJU69121.1, AJU68429.1, AJU67713.1, AJU66996.1, AJU66318.1, AJU65601.1,AJU64885.1, AJU64173.1, AJU63483.1, AJU62784.1, AJU62085.1, AJU61373.1,AJU60685.1, AJU60022.1, AJU59308.1, AJU58621.1, AJU57919.1, AJP37799.1,AHY75069.1, EGW34937.1, ABN67480.2, XP_007372349.1, EWG97048.1,ACB46526.1, XP_001385509.2, ACY82596.1, ACY82595.1, EEU05123.1,EDN60419.1, DAA05785.1, CAC17748.1, XP_013021409.1, XP_013017766.1,KMK60772.1, EPY53152.1, EPX75323.1 GI:528065761, EEB06603.1,XP_002172896.1, KEY78745.1, EXX70387.1, EXX62686.1, EXX62685.1,EXX62684.1, EXX62683.1, GAA85661.1, XP_755036.1 or CAD24957.1.

Various methods are known to those skilled in the art to impede orprevent the expression of the endogenous tps2 gene or the endogenoustps2 gene ortholog. In the context of the present disclosure, a genewhich is “endogenous” to a yeast is understood to mean that such gene isnatively provided in the organism. For example, a gene encoding a TPS2polypeptide having phosphatase activity is considered endogenous toyeast has been natively produced by such yeast and is not the result ofan in vitro genetic modification. As an another example, a gene isconsidered to be endogenous to a yeast is considered to have beennatively included in or produced by such yeast and was not deliberatelyintroduced by genetic means in the yeast.

In order to impede or prevent the expression of the tps2 gene or itsortholog, the recombinant yeast strain can be genetically engineered todisrupt the open-reading frame of the endogenous tps2 gene or itsortholog by inserting a non-coding sequence or adding one or morenucleic acid residues from the open-reading frame. In such instance,while a section of the tps2 gene or of the tps2 gene ortholog could beexpressed (for example, the section of the gene which precedes theinsertion or addition) but a functional TPS2 polypeptide could not beproduced (due to the presence of a non-coding sequence or an additioninterrupting the translation of the full-length TPS2 polypeptide).Alternatively (or in combination), the recombinant yeast strain can begenetically-engineered to remove a part or the totality of theendogenous tps2 gene or ortholog from the yeast's genome. In the contextof the present disclosure, a deletion refers to the removal of at leastone nucleic acid residue of the tps2 gene. In such instance, while asection of the tps2 gene or its ortholog could be expressed (for examplethe section (if any) which precedes the deletion) but a functional TPS2polypeptide could not be produced. In another alternative, therecombinant yeast strain can be genetically-engineered to include one ofmore nucleic acid residue substitution in the tps2 gene or in the tps2gene ortholog. The one or more nucleic acid residue substitution cancause the introduction of a stop codon in the open-reading frame of thetps2 gene/ortholog or at least one amino acid substitution in thecorresponding polypeptide which will no longer be considered afunctional or biologically active TPS2 polypeptide. The recombinantyeast strain can be genetically engineered to impede or prevent theexpression of the tps2 gene or its ortholog by manipulating thenon-coding sequence (promoter for example) associated with the codingsequence of the tps2 gene. The nucleic acid sequence of the promoter ofthe tps2 gene can be modified to remove, add and/or substitute at leastone nucleic acid residue so as to reduce or prevent the expression ofthe tps2 gene or its ortholog. The mutation, disruption and/or deletioncan be made in one of the copy of the tps2 gene or its ortholog presentin the yeast's genome or in both copies of the tps2 gene or its orthologpresent in the yeast's genome.

The second genetic modification can be associated with the production ofan heterologous trehalase, a trehalase variant or a trehalase fragment(having trehalase activity). In such instance, the genetic manipulationis made to add of an heterologous trehalase-encoding gene (and,optionally, additional non-coding region for facilitating or increasingthe expression of the trehalase-encoding gene) and is intended to eitherprovide or increase trehalase activity of the recombinant strain.

As used in the context of the present disclosure, a trehalase is anenzyme capable of hydrolyzing one molecule of trehalose in two moleculesof glucose. Trehalases (α,α-trehalose-1-C-glucohydrolase, EC 3.2.1.28)have been reported from many organisms including prokaryotes, plants andanimals. At least two-types of trehalases, based on their pH optima,have been characterized: acid trehalases (mostly extracellular, usuallyassociated with the yeast's membrane) and neutral trehalases (usuallycytosolic). The recombinant yeast strain of the present disclosure canbe genetically engineered to express an acid trehalase, a neutraltrehalase or both. In some instances, the heterologous trehalase isproduced and transported outside the yeast cell (e.g., extracellular).

The heterologous trehalase(s) expressed by the recombinant yeast straincan be provided from any heterologous organism (yeast, bacteria, plantsor animals). The term “heterologous” when used in reference to a nucleicacid molecule (such as a promoter or a coding sequence) or a trehalaserefers to a nucleic acid molecule or a trehalase that is not nativelyfound in the host yeast. “Heterologous” also includes a native codingregion, or portion thereof, that is removed from the source organism andsubsequently reintroduced into the source organism in a form that isdifferent from the corresponding native gene, e.g., not in its naturallocation in the organism's genome. The heterologous nucleic acidmolecule is purposively introduced into the yeast. A “heterologous”nucleic acid molecule or trehalase may be derived from any source, e.g.,eukaryotes, prokaryotes, viruses, etc. In an embodiment, theheterologous nucleic acid molecule may be derived from an eukaryote(such as, for example, another yeast) or a prokaryote (such as, forexample, a bacteria). The term “heterologous” as used herein also refersto an element (nucleic acid or protein) that is derived from a sourceother than the endogenous source. Thus, for example, a heterologouselement could be derived from a different strain of host cell, or froman organism of a different taxonomic group (e.g., different kingdom,phylum, class, order, family genus, or species, or any subgroup withinone of these classifications). The term “heterologous” is also usedsynonymously herein with the term “exogenous”.

The heterologous trehalase can be derived from the genus Aspergillusand, in some instances, from the species Aspergillus fumigatus orAspergillus nidulans. It is possible to use an heterologous trehalasewhich does not comprise a tethering region and does not have the abilityto associate with the surface of the cell producing same. In someembodiments, the heterologous trehalase has or consists of the aminoacid sequence of SEQ ID NO: 1 or 2. For example, the recombinant yeasthost cell can be genetically manipulated to express one or moreheterologous trehalase genes.

The heterologous trehalase can be a variant of a known trehalase, forexample a variant of the trehalase having the amino acid sequence of SEQID NO: 1 or 2. The trehalase variants have at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to thetrehalases described herein. A variant comprises at least one amino aciddifference when compared to the amino acid sequence of the nativetrehalase. The term “percent identity”, as known in the art, is arelationship between two or more polypeptide sequences or two or morepolynucleotide sequences, as determined by comparing the sequences. Thelevel of identity can be determined conventionally using known computerprograms. Identity can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, N Y (1988); Biocomputing:Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, N Y(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., andGriffin, H. G., eds.) Humana Press, N J (1994); Sequence Analysis inMolecular Biology (von Heinje, G., ed.) Academic Press (1987); andSequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) StocktonPress, NY (1991). Preferred methods to determine identity are designedto give the best match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Sequence alignments and percent identity calculationsmay be performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignments of the sequences disclosed herein were performed using theClustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)with the default parameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y=10).Default parameters for pairwise alignments using the Clustal method wereKTUPLB 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The variant heterologous trehalases described herein may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide for purification of the polypeptide.

A “variant” of the trehalase can be a conservative variant or an allelicvariant. As used herein, a conservative variant refers to alterations inthe amino acid sequence that do not adversely affect the biologicalfunctions of the trehalase. A substitution, insertion or deletion issaid to adversely affect the protein when the altered sequence preventsor disrupts a biological function associated with the trehalase (e.g.,the hydrolysis of trehalose into two glucose molecules). For example,the overall charge, structure or hydrophobic-hydrophilic properties ofthe protein can be altered without adversely affecting a biologicalactivity. Accordingly, the amino acid sequence can be altered, forexample to render the peptide more hydrophobic or hydrophilic, withoutadversely affecting the biological activities of the trehalase.

The heterologous trehalase can be a fragment of a known trehalase orfragment of a variant of a known trehalase (such as, for example, afragment of the trehalase having the amino acid sequence of SEQ ID NO: 1or 2). Trehalase “fragments” have at least at least 100, 200, 300, 400,500, 600, 700, 800, 900, 1 000 or more consecutive amino acids of thetrehalase. A fragment comprises at least one less amino acid residuewhen compared to the amino acid sequence of the trehalase and stillpossess the enzymatic activity of the full-length trehalase. In someembodiments, fragments of the trehalases can be employed for producingthe corresponding full-length trehalase by peptide synthesis. Therefore,the fragments can be employed as intermediates for producing thefull-length proteins.

The heterologous nucleic acid molecule encoding the heterologoustrehalase, variant or fragment can be integrated in the genome of theyeast host cell. The term “integrated” as used herein refers to geneticelements that are placed, through molecular biology techniques, into thegenome of a host cell. For example, genetic elements can be placed intothe chromosomes of the host cell as opposed to in a vector such as aplasmid carried by the host cell. Methods for integrating geneticelements into the genome of a host cell are well known in the art andinclude homologous recombination. The heterologous nucleic acid moleculecan be present in one or more copies in the yeast host cell's genome.Alternatively, the heterologous nucleic acid molecule can beindependently replicating from the yeast's genome. In such embodiment,the nucleic acid molecule can be stable and self-replicating.

The present disclosure also provides nucleic acid molecules formodifying the yeast host cell so as to allow the expression of theheterologous trehalase, variant or fragment. The nucleic acid moleculemay be DNA (such as complementary DNA, synthetic DNA or genomic DNA) orRNA (which includes synthetic RNA) and can be provided in a singlestranded (in either the sense or the antisense strand) or a doublestranded form. The contemplated nucleic acid molecules can includealterations in the coding regions, non-coding regions, or both. Examplesare nucleic acid molecule variants containing alterations which producesilent substitutions, additions, or deletions, but do not alter theproperties or activities of the encoded trehalases, variants orfragments.

In some embodiments, the nucleic acid molecules encoding theheterologous trehalase and/or glucoamylase, fragment or variant arecodon-optimized with respect to the intended recipient recombinant yeasthost cell. As used herein the term “codon-optimized coding region” meansa nucleic acid coding region that has been adapted for expression in thecells of a given organism by replacing at least one, or more than one,codons with one or more codons that are more frequently used in thegenes of that organism. In general, highly expressed genes in anorganism are biased towards codons that are recognized by the mostabundant tRNA species in that organism. One measure of this bias is the“codon adaptation index” or “CAI,” which measures the extent to whichthe codons used to encode each amino acid in a particular gene are thosewhich occur most frequently in a reference set of highly expressed genesfrom an organism. The CAI of codon optimized sequences described hereincorresponds to between about 0.8 and 1.0, between about 0.8 and 0.9, orabout 1.0.

The present disclosure also provides nucleic acid molecules that arehybridizable to the complement nucleic acid molecules encoding theheterologous trehalase, the heterologous glucoamylase as well asvariants or fragments. A nucleic acid molecule is “hybridizable” toanother nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, whena single stranded form of the nucleic acid molecule can anneal to theother nucleic acid molecule under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified, e.g., in Sambrook, J.,Fritsch, E. F. and Maniatis, T. MOLECULAR CLONING: A LABORATORY MANUAL,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(1989), particularly Chapter 11 and Table 11.1 therein. The conditionsof temperature and ionic strength determine the “stringency” of thehybridization. Stringency conditions can be adjusted to screen formoderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofconditions uses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. For more stringent conditions, washes are performed at highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS areincreased to 60° C. Another set of highly stringent conditions uses twofinal washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of highlystringent conditions are defined by hybridization at 0.1×SSC, 0.1% SDS,65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS.

Hybridization requires that the two nucleic acid molecules containcomplementary sequences, although depending on the stringency of thehybridization, mismatches between bases are possible. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation, variables well known inthe art. The greater the degree of similarity or homology between twonucleotide sequences, the greater the value of Tm for hybrids of nucleicacids having those sequences. The relative stability (corresponding tohigher Tm) of nucleic acid hybridizations decreases in the followingorder: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100nucleotides in length, equations for calculating Tm have been derived.For hybridizations with shorter nucleic acids, i.e. e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity. In oneembodiment the length for a hybridizable nucleic acid is at least about10 nucleotides. Preferably a minimum length for a hybridizable nucleicacid is at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least 30 nucleotides.Furthermore, the skilled artisan will recognize that the temperature andwash solution salt concentration may be adjusted as necessary accordingto factors such as length of the probe.

The nucleic acid molecules comprise a coding region for the heterologoustrehalase as well as its variants and fragments. A DNA or RNA “codingregion” is a DNA or RNA molecule which is transcribed and/or translatedinto a polypeptide in a cell in vitro or in vivo when placed under thecontrol of appropriate regulatory sequences. “Suitable regulatoryregions” refer to nucleic acid regions located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingregion, and which influence the transcription, RNA processing orstability, or translation of the associated coding region. Regulatoryregions may include promoters, translation leader sequences, RNAprocessing site, effector binding site and stem-loop structure. Theboundaries of the coding region are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding region can include, but is not limited to,prokaryotic regions, cDNA from mRNA, genomic DNA molecules, syntheticDNA molecules, or RNA molecules. If the coding region is intended forexpression in a eukaryotic cell, a polyadenylation signal andtranscription termination sequence will usually be located 3′ to thecoding region. In an embodiment, the coding region can be referred to asan open reading frame. “Open reading frame” is abbreviated ORF and meansa length of nucleic acid, either DNA, cDNA or RNA, that comprises atranslation start signal or initiation codon, such as an ATG or AUG, anda termination codon and can be potentially translated into a polypeptidesequence.

The nucleic acid molecules described herein can comprise transcriptionaland/or translational control regions. “Transcriptional and translationalcontrol regions” are DNA regulatory regions, such as promoters,enhancers, terminators, and the like, that provide for the expression ofa coding region in a host cell. In eukaryotic cells, polyadenylationsignals are control regions.

The heterologous nucleic acid molecule can be introduced in the yeasthost cell using a vector. A “vector,” e.g., a “plasmid”, “cosmid” or“YAC” (yeast artificial chromosome) refers to an extra chromosomalelement and is usually in the form of a circular double-stranded DNAmolecule. Such vectors may be autonomously replicating sequences, genomeintegrating sequences, phage or nucleotide sequences, linear, circular,or supercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

In the heterologous nucleic acid molecule, the promoter and the nucleicacid molecule(s) coding for the heterologous protein(s) are operativelylinked to one another. In the context of the present disclosure, theexpressions “operatively linked” or “operatively associated” refers tofact that the promoter is physically associated to the nucleotide acidmolecule coding for the heterologous protein in a manner that allows,under certain conditions, for expression of the heterologous proteinfrom the nucleic acid molecule. In an embodiment, the promoter can belocated upstream (5′) of the nucleic acid sequence coding for theheterologous protein. In still another embodiment, the promoter can belocated downstream (3′) of the nucleic acid sequence coding for theheterologous protein. In the context of the present disclosure, one ormore than one promoter can be included in the heterologous nucleic acidmolecule. When more than one promoter is included in the heterologousnucleic acid molecule, each of the promoters is operatively linked tothe nucleic acid sequence coding for the heterologous protein. Thepromoters can be located, in view of the nucleic acid molecule codingfor the heterologous protein, upstream, downstream as well as bothupstream and downstream.

“Promoter” refers to a DNA fragment capable of controlling theexpression of a coding sequence or functional RNA. The term“expression,” as used herein, refers to the transcription and stableaccumulation of sense (mRNA) from the heterologous nucleic acid moleculedescribed herein. Expression may also refer to translation of mRNA intoa polypeptide. Promoters may be derived in their entirety from a nativegene, or be composed of different elements derived from differentpromoters found in nature, or even comprise synthetic DNA segments. Itis understood by those skilled in the art that different promoters maydirect the expression at different stages of development, or in responseto different environmental or physiological conditions. Promoters whichcause a gene to be expressed in most cells at most times at asubstantial similar level are commonly referred to as “constitutivepromoters”. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths may have identical promoter activity. Apromoter is generally bounded at its 3′ terminus by the transcriptioninitiation site and extends upstream (5′ direction) to include theminimum number of bases or elements necessary to initiate transcriptionat levels detectable above background. Within the promoter will be founda transcription initiation site (conveniently defined for example, bymapping with nuclease S1), as well as protein binding domains (consensussequences) responsible for the binding of the polymerase.

The promoter can be heterologous to the nucleic acid molecule encodingthe heterologous protein. The promoter can be heterologous or derivedfrom a strain being from the same genus or species as the recombinantyeast host cell. In an embodiment, the promoter is derived from the samegenera or species of the yeast host cell and the heterologous protein isderived from different genera that the yeast host cell.

In the context of the present disclosure, the heterologous protein canbe further modified to include a tethering region (so as to allow thelocalization of the secreted heterologous protein at the externalsurface of the yeast host cell) and/or fused to another entity (tocreate a fusion protein). Alternatively, the heterologous protein (suchas the heterologous trehalase) can be modified so as to remove itstethering region.

In the context of the present disclosure, the recombinant yeast hostcell can include at least two “second” genetic modifications, one inleading to the reduction in the production of one or more native enzymesthat function to produce trehalose or regulating trehalose synthesis andanother one leading to the expression of an heterologous trehalase.

In some instances, the recombinant yeast host cell can include a furthergenetic modification for reducing the production of one or more nativeenzyme that function to catabolize (breakdown) formate. As used in thecontext of the present disclosure, the expression “native polypeptidesthat function to catabolize formate” refers to polypeptides which areendogenously found in the recombinant yeast host cell. Native enzymesthat function to catabolize formate include, but are not limited to, theFDH1 and the FDH2 polypeptides (also referred to as FDH1 and FDH2respectively). In an embodiment, the recombinant yeast host cell bears agenetic modification in at least one of the fdh1 gene (encoding the FDH1polypeptide), the fdh2 gene (encoding the FDH2 polypeptide) or orthologsthereof. In another embodiment, the recombinant yeast host cell bearsgenetic modifications in both the fdh1 gene (encoding the FDH1polypeptide) and the fdh2 gene (encoding the FDH2 polypeptide) ororthologs thereof. Examples of recombinant yeast host cells bearing suchgenetic modification(s) leading to the reduction in the production ofone or more native enzymes that function to catabolize formate aredescribed in WO 2012/138942. Preferably, the recombinant yeast host cellhas genetic modifications (such as a genetic deletion or insertion) inthe fdh1 gene and in the fdh2 gene which would cause the host cell tohave knocked-out fdh1 and fdh2 genes.

In some instances, the recombinant yeast host cell can include a furthergenetic modification allowing the expression of an heterologousglucoamylase. In an embodiment, the heterologous glucoamylase is derivedfrom a γ-amylase, such as, for example, the glucoamylase ofSaccharomycoces filbuligera (e.g., encoded by the glu 0111 gene). Ininstances in which the recombinant yeast host cell is intended to beused at elevated temperatures, genetic modifications for increasing therobustness of a genetically-modified recombinant yeast host cellexpressing an heterologous glucoamylase are described inPCT/IB2016/055162 filed on Aug. 29, 2016 and herewith incorporated inits entirety.

The recombinant yeast host cell can be further genetically modified toallow for the production of additional heterologous proteins. In anembodiment, the recombinant yeast host cell can be used for theproduction of an enzyme, and especially an enzyme involved in thecleavage or hydrolysis of its substrate (e.g., a lytic enzyme and, insome embodiments, a saccharolytic enzyme). In still another embodiment,the enzyme can be a glycoside hydrolase. In the context of the presentdisclosure, the term “glycoside hydrolase” refers to an enzyme involvedin carbohydrate digestion, metabolism and/or hydrolysis, includingamylases, cellulases, hemicellulases, cellulolytic and amylolyticaccessory enzymes, inulinases, levanases, trehalases, pectinases, andpentose sugar utilizing enzymes. In another embodiment, the enzyme canbe a protease. In the context of the present disclosure, the term“protease” refers to an enzyme involved in protein digestion, metabolismand/or hydrolysis. In yet another embodiment, the enzyme can be anesterase. In the context of the present disclosure, the term “esterase”refers to an enzyme involved in the hydrolysis of an ester from an acidor an alcohol, including phosphatases such as phytases.

The additional heterologous protein can be an “amylolytic enzyme”, anenzyme involved in amylase digestion, metabolism and/or hydrolysis. Theterm “amylase” refers to an enzyme that breaks starch down into sugar.All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds.Some amylases, such as γ-amylase (glucoamylase), also act onα-1,6-glycosidic bonds. Amylase enzymes include α-amylase (EC 3.2.1.1),β-amylase (EC 3.2.1.2), and γ-amylase (EC 3.2.1.3). The α-amylases arecalcium metalloenzymes, unable to function in the absence of calcium. Byacting at random locations along the starch chain, α-amylase breaks downlong-chain carbohydrates, ultimately yielding maltotriose and maltosefrom amylose, or maltose, glucose and “limit dextrin” from amylopectin.Because it can act anywhere on the substrate, α-amylase tends to befaster-acting than β-amylase. In an embodiment, the heterologous proteinis derived from a α-amylase such as, for example, from the α-amylase ofBacillus amyloliquefacens. Another form of amylase, β-amylase is alsosynthesized by bacteria, fungi, and plants. Working from thenon-reducing end, β-amylase catalyzes the hydrolysis of the second α-1,4glycosidic bond, cleaving off two glucose units (maltose) at a time.Another amylolytic enzyme is α-glucosidase that acts on maltose andother short malto-oligosaccharides produced by α-, β-, and γ-amylases,converting them to glucose. Another amylolytic enzyme is pullulanase.Pullulanase is a specific kind of glucanase, an amylolytic exoenzyme,that degrades pullulan. Pullulan is regarded as a chain of maltotrioseunits linked by alpha-1,6-glycosidic bonds. Pullulanase (EC 3.2.1.41) isalso known as pullulan-6-glucanohydrolase (debranching enzyme). Anotheramylolytic enzyme, isopullulanase, hydrolyses pullulan to isopanose(6-alpha-maltosylglucose). Isopullulanase (EC 3.2.1.57) is also known aspullulan 4-glucanohydrolase. An “amylase” can be any enzyme involved inamylase digestion, metabolism and/or hydrolysis, including α-amylase,β-amylase, glucoamylase, pullulanase, isopullulanase, andalpha-glucosidase.

The additional heterologous protein can be a “cellulolytic enzyme”, anenzyme involved in cellulose digestion, metabolism and/or hydrolysis.The term “cellulase” refers to a class of enzymes that catalyzecellulolysis (i.e. the hydrolysis) of cellulose. Several different kindsof cellulases are known, which differ structurally and mechanistically.There are general types of cellulases based on the type of reactioncatalyzed: endocellulase breaks internal bonds to disrupt thecrystalline structure of cellulose and expose individual cellulosepolysaccharide chains; exocellulase cleaves 2-4 units from the ends ofthe exposed chains produced by endocellulase, resulting in thetetrasaccharides or disaccharide such as cellobiose. There are two maintypes of exocellulases (or cellobiohydrolases, abbreviate CBH)—one typeworking processively from the reducing end, and one type workingprocessively from the non-reducing end of cellulose; cellobiase orbeta-glucosidase hydrolyses the exocellulase product into individualmonosaccharides; oxidative cellulases that depolymerize cellulose byradical reactions, as for instance cellobiose dehydrogenase (acceptor);cellulose phosphorylases that depolymerize cellulose using phosphatesinstead of water. In the most familiar case of cellulase activity, theenzyme complex breaks down cellulose to beta-glucose. A “cellulase” canbe any enzyme involved in cellulose digestion, metabolism and/orhydrolysis, including an endoglucanase, glucosidase, cellobiohydrolase,xylanase, glucanase, xylosidase, xylan esterase, arabinofuranosidase,galactosidase, cellobiose phosphorylase, cellodextrin phosphorylase,mannanase, mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, and feruoyl esterase protein.

The additional heterologous protein can have “hemicellulolyticactivity”, an enzyme involved in hemicellulose digestion, metabolismand/or hydrolysis. The term “hemicellulase” refers to a class of enzymesthat catalyze the hydrolysis of cellulose. Several different kinds ofenzymes are known to have hemicellulolytic activity including, but notlimited to, xylanases and mannanases.

The additional heterologous protein can have “xylanolytic activity”, anenzyme having the is ability to hydrolyze glycosidic linkages inoligopentoses and polypentoses. The term “xylanase” is the name given toa class of enzymes which degrade the linear polysaccharidebeta-1,4-xylan into xylose, thus breaking down hemicellulose, one of themajor components of plant cell walls. Xylanases include those enzymesthat correspond to Enzyme Commission Number 3.2.1.8. The heterologousprotein can also be a “xylose metabolizing enzyme”, an enzyme involvedin xylose digestion, metabolism and/or hydrolysis, including a xyloseisomerase, xylulokinase, xylose reductase, xylose dehydrogenase, xylitoldehydrogenase, xylonate dehydratase, xylose transketolase, and a xylosetransaldolase protein. A “pentose sugar utilizing enzyme” can be anyenzyme involved in pentose sugar digestion, metabolism and/orhydrolysis, including xylanase, arabinase, arabinoxylanase,arabinosidase, arabinofuranosidase, arabinoxylanase, arabinosidase, andarabinofuranosidase, arabinose isomerase, ribulose-5-phosphate4-epimerase, xylose isomerase, xylulokinase, xylose reductase, xylosedehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylosetransketolase, and/or xylose transaldolase.

The additional heterologous protein can have “mannanic activity”, anenzyme having the is ability to hydrolyze the terminal, non-reducingR-D-mannose residues in β-D-mannosides. Mannanases are capable ofbreaking down hemicellulose, one of the major components of plant cellwalls. Xylanases include those enzymes that correspond to EnzymeCommission Number 3.2.25.

The additional heterologous protein can be a “pectinase”, an enzyme,such as pectolyase, pectozyme and polygalacturonase, commonly referredto in brewing as pectic enzymes. These enzymes break down pectin, apolysaccharide substrate that is found in the cell walls of plants.

The additional heterologous protein can have “phytolytic activity”, anenzyme catalyzing the conversion of phytic acid into inorganicphosphorus. Phytases (EC 3.2.3) can be belong to the histidine acidphosphatases, β-propeller phytases, purple acid phosphastases or proteintyrosine phosphatase-like phytases family.

The additional heterologous protein can have “proteolytic activity”, anenzyme involved in protein digestion, metabolism and/or hydrolysis,including serine proteases, threonine proteases, cysteine proteases,aspartate proteases, glutamic acid proteases and metalloproteases.

When the recombinant yeast host cell expresses an heterologous protein,it can be further modified to increase its robustness at hightemperatures. Genetic modifications for increasing the robustness of agenetically-modified recombinant yeast host cell are described in U.S.62/214,412 filed on Sep. 4, 2015 and herewith incorporated in itsentirety.

Methods of Using the Recombinant Yeast Host Cells for Limiting theAccumulation of Trehalose During Fermentation

The recombinant yeast host cells described herein can be used to limitand, in some embodiments, prevent the production, accumulation orexcretion of trehalose during fermentation. As indicated above, therecombinant yeast host cells have a second genetic modification whicheither limits the production of endogenous trehalose by the recombinantyeast or hydrolyzes the trehalose that is being endogenously production.

The process comprises combining a substrate to be hydrolyzed (optionallyincluded in a fermentation medium) with the recombinant yeast hostcells. In an embodiment, the substrate to be hydrolyzed is alignocellulosic biomass and, in some embodiments, it comprises starch(in a gelatinized or raw form). In other embodiments, the substrate tobe hydrolyzed comprises maltodextrin. In some embodiments, the use ofrecombinant yeast host cells limits or avoids the need of addingtrehalase in a purified form during fermentation to limit the amount oftrehalose. This embodiment is advantageous because it can reduce oreliminate the need to supplement the fermentation medium with externalsource of purified enzymes (e.g., glucoamylase and/or trehalase) whileallowing the fermentation of the lignocellulosic biomass into afermentation product (such as ethanol). However, in some circumstances,it may be advisable to supplement the medium with a trehalase (such as,for example, the trehalase having the amino acid sequence of SEQ ID NO:1 or 2) in a purified. Such trehalase can be produced in a recombinantfashion in a recombinant yeast host cell.

The recombinant yeast host cells described herein can be used toincrease the production of a fermentation product during fermentation.As indicated above, the recombinant yeast host cells have a secondgenetic modification which either limits the production of endogenoustrehalose by the recombinant yeast or hydrolyzes the trehalose that isbeing endogenously production and such second genetic modifications canimprove the yield in one or more fermentation products. The processcomprises combining a substrate to be hydrolyzed (optionally included ina fermentation medium) with the recombinant yeast host cells. In anembodiment, the substrate to be hydrolyzed is a lignocellulosic biomassand, in some embodiments, it comprises starch (in a gelatinized or rawform). In some embodiments, the use of recombinant yeast host cellslimits or avoids the need of adding trehalase in a purified form duringfermentation to limit the amount of trehalose. This embodiment isadvantageous because it can reduce or eliminate the need to supplementthe fermentation medium with external source of purified enzymes (e.g.,glucoamylase and/or trehalase) while allowing the fermentation of thelignocellulosic biomass into a fermentation product (such as ethanol).However, in some circumstances, it may be advisable to supplement themedium with a trehalase (such as, for example, the trehalase having theamino acid sequence of SEQ ID NO: 1 or 2) in a purified. Such trehalasecan be produced in a recombinant fashion in a recombinant yeast hostcell.

The production of ethanol can be performed at temperatures of at leastabout 25° C., about 28° C. about 30° C. about 31° C. about 32° C. about33° C., about 34° C., about 35° C., about 36° C., about 37° C., about38° C., about 39° C., about 40° C., about 41° C., about 42° C., or about50° C. In some embodiments, when a thermotolerant yeast cell is used inthe process, the process can be conducted at temperatures above about30° C., about 31° C., about 32° C., about 33° C., about 34° C., about35° C., about 36° C., about 37° C., about 38° C., about 39° C., about40° C., about 41° C., about 42° C., or about 50° C.

In some embodiments, the process can be used to produce ethanol at aparticular rate. For example, in some embodiments, ethanol is producedat a rate of at least about 0.1 mg per hour per liter, at least about0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, atleast about 0.75 mg per hour per liter, at least about 1.0 mg per hourper liter, at least about 2.0 mg per hour per liter, at least about 5.0mg per hour per liter, at least about 10 mg per hour per liter, at leastabout 15 mg per hour per liter, at least about 20.0 mg per hour perliter, at least about 25 mg per hour per liter, at least about 30 mg perhour per liter, at least about 50 mg per hour per liter, at least about100 mg per hour per liter, at least about 200 mg per hour per liter, orat least about 500 mg per hour per liter.

Ethanol production can be measured using any method known in the art.For example, the quantity of ethanol in fermentation samples can beassessed using HPLC analysis. Many ethanol assay kits are commerciallyavailable that use, for example, alcohol oxidase enzyme based assays.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example I—Material and Methods

Strain M4652 was constructed using the KT (Kanamycin and HSV-thymidinekinase) and NT (nourseothricin and HSV-thymidine kinase) recyclable MXcassettes (FIG. 8A) targeting a direct integration and removal of thetps1 open reading frame. The KT-max and NT-max cassettes were PCRamplified, along with non-coding 5′ and 3′ flanks, creating overlappinghomologous ends to promote recombination in vivo. The PCR products weretransformed into the diploid Saccharomyces cerevisiae host strain,M2390, and subsequently selected on YPD containing G418 (200 μg/ml) andcloNat (100 μg/ml) to select for removal of both tps1 alleles. Table 1below provides the nucleic acid sequence of the primers used to make theMA613 genetic cassette used to create the M4652 strain.

TABLE 1 Nucleic acid sequence of the primersused to make the MA613 genetic cassette used to create the M4652 strain. SEQ ID  Target NO: SequenceTPS1 5′ 19 GCAGAGGATTACTT Flank GGACATTAACGGTT CTCCTATC 20GGACGAGGCAAGCTA AACAGATCTCTAGAC CTAAGTTCTATGTCT TAATAAGTCTGTATG KT-MX 21 TACTCACATACAGAC and TTATTAAGACATAGA NT-MX ACTTAGGTCTAGAGATCTGTTTAGCTTGCC 22 GAATAGACGATCGTC TCATTTGCATCGGGT TCAGAGACTACATGATAGTCCAAAGAAAAG TPS2 3′ 23 CCGTTTCTTTTCTTT Flank GGACTATCATGTAGTCTCTGAACCCGATGC AAATGAGACGATCGT 24 GCAAGAGGCTCCTCC ACTGGCATTTTCACGATTTGG

Strain M4653 was constructed using the same method as described in theM4652 engineering. However, the 5′ and 3′ non-coding flanking regionswere designed to target the tps2 region (FIG. 81) for the deletion ofthe tps2 open reading frame. Table 2 below provides the nucleic acidsequence of the primers used to make the MA614 genetic cassette used tocreate the M4653 strain.

TABLE 2  Nucleic acid sequence of the primersused to make the MA614 genetic cassette used to create the M4653 strain.SEQ ID Target NO: Sequence TPS2 5′ 25 GCTGTGCAGCAG Flank GGTATTCTACTACGTGTTAGCTT 26 GGACGAGGCAAG CTAAACAGATCT CTAGACCTATTC GGCACAGAAATAGTGACAGGCAGT KT-MX and 27 AATAACACTGCC NT-MX TGTCACTATTTC TGTGCCGAATAGGTCTAGAGATCT GTTTAGCTTGCC 28 TCTAGTCATAAC CATTTCGTTAAA AAGGGTGTTGAGACTACATGATAG TCCAAAGAAAAG TPS2 3′ 29 CCGTTTCTTTTC Flank TTTGGACTATCATGTAGTCTCAAC ACCCTTTTTAAC GAAATGGTTATG

Strain M11245 was engineered to express an heterologous trehalase. Theheterologous trehalase gene, MP244 (SEQ ID NO: 1) was codon-optimizedfor S. cerevisiae based on the amino acid sequence from Aspergillusfumigatus (GenBank Accession No. XP_748551). The synthesized sequencewas used as PCR template to create homologous ends with the S.cerevisiae tef2 promoter and adh3 terminator and integrated at then FCY1loci in the diploid S. cerevisiae host strain via homologousrecombination in vivo (FIG. 8). Table 3 below provides the nucleic acidsequence of the primers used to make the MA1920 genetic cassette used tocreate the M11245 strain.

TABLE 3  Nucleic acid sequence of the primers used to make the MA1920 genetic cassetteused to create the M11245 strain. SEQ  ID Target NO: Sequence FCY1 5′ 31CTCGTTGGTAGG Flank GTCCACACCATA GACTTCAG 32 TAGCTATGAAAT TTTTAACTCTTTAAGCTGGCTCT TEF2p 33 GATGAGAGCCAG CTTAAAGAGTTA AAAATTTCATAG CTAGGGCGCCATAACCAAGGTATC 34 CCAACAAAGAAA CCCAAGTAGCCA AGTTTTGAGACA ACATGTTTAGTTAATTATAGTTCG MP244 35 GAATATACGGTC AACGAACTATAA TTAACTAAACATGTTGTCTCAAAA CTTGGC 36 CAAAGACTTTCA TAAAAAGTTTGG GTGCGTAACACGCTATCAAGCGTT GAATTGTCTG ADH3t 37 GCTTTGAACGAC AGAAGATACAGA CAATTCAACGCTTGATAGCGTGTT ACGCACCCAAAC 38 TATATAAAATTA AATACGTAAATA CAGCGTGCTGCGTGCTATGAGGAA GAAATCCAAATC FCY1 3′ 39 AGCACGCAGCAC Flank GCTGTATTTACGTATTTAATTTT 40 GTAGTGCTGTCT GAACAGAATAAA TGCGTTCTTGG

Strain M10957 was engineered to express an heterologous trehalase. Theheterologous trehalase gene, MP848 (SEQ ID NO: 2) was codon-optimizedfor S. cerevisiae based on the amino acid sequence from Aspergillusnidulans (GenBank Accession No. P78617). The synthesized sequence wasused as PCR template to create homologous ends with the S. cerevisiaetef2 promoter and adh3 terminator and integrated at the FCY1 loci in thediploid S. cerevisiae host strain via homologous recombination in vivo(FIG. 8). Table 4 below provides the nucleic acid sequence of theprimers used to make the MAP516 genetic cassette used to create theM10957 strain.

TABLE 4 Nucleic acid sequence of the primersused to make the MAP516 genetic cassette used to createthe M10957 strain. SEQ ID Target NO: Sequence FCY1 5′ 41 CTGACTCGTTGGTFlank GGGGTCCACACCA TAGA 42 GATTGGCGGTCTA TAGATACCTTGGT TATGGCGCCCTAGCTATGAAATTTTT AACTCTTC TEF2p 43 GAGAGCCAGCTTT TTGAAGAGTTAAAAATTTCATAGCTA GGGCGCCATAACC AAGGTATC 44 GTTTAGTTAATTA TAGTTCG MP848 45TTTTTAGAATATA CGGTCAACGAACT ATAATTAACTAAA CATGAGATTCAAG TCCGTTTT 46AATGAAAAAAAAA GTGGTAGATTGGG CTACGTAAATTCG ATTACAACAAAGG AACTGGTT ADH3t47 TCGAATTTACGTA GCCCAATC 48 TATATAAAATTAA ATACGTAAATACA GCGTGCTGCGTGCTCAAATGACGTCA AAAGAAGT FCY1 3′ 49 CATAGGCTCATAT Flank AATACTTCTTTTGACGTCATTTGAGC ACGCAGCACGCTG TATTTACG 50 GTAGTGCTGTCTG AACAGAATAAATGCGTTCT

Strain M12121 was constructed using the M11589 background containing theglycerol reduction pathway and heterologous glu011-CO glucoamylase Thesynthesized sequence was used as PCR template to create homologous endswith the S. cerevisiae tef2 promoter and adh3 terminator and integratedat the IME loci in the diploid S. cerevisiae host strain via homologousrecombination in vivo (FIG. 80). Table 5 below provides the nucleic acidsequence of the primers used to make the MAP811 genetic cassette used tocreate the M12121 strain.

TABLE 5 Nucleic acid sequence of the primersused to make the MAP811 genetic cassette used to create theM12121 strain. SEQ ID Target NO: Sequence IME1 5′ 51 CACCTACAGAGAAAFlank CAAATTCCTACTGG CACCC 52 TTGGCGGTCTATAG ATACCTTGGTTATGGCGCCCGTCGACAA CTAAACTGGAATGT GAGG TEF2p 53 ACTTTTGTTGTTCCCTCACATTCCAGTT TAGTTGTCGACGGG CGCCATAACCAAGG TATC 54 CCAACAAAGAAACCCAAGTAGCCAAGTT TTGAGACAACATGT TTAGTTAATTATAG TTCG MP244 55GAATATACGGTCAA CGAACTATAATTAA CTAAACATGTTGTC TCAAAACTTGGCTA CTTG 56AAATGAAAAAAAAA GTGGTAGATTGGGC TACGTAAATTCGAT CAAGCGTTGAATTG TCTG IDP1t57 GCTTTGAACGACAG AAGATACAGACAAT TCAACGCTTGATCG AATTTACGTAGCCC AATC 58ATTTTGAGGGAAGG GGGAAGATTGTAGT ACTTTTCGAGAACA AATGACGTCAAAAG AAGT IME1 3′59 TAGGCTCATATAAT Flank ACTTCTTTTGACGT CATTTGTTCTCGAA AAGTACTACAATCTTCCC 60 GAACTTCTGCCTTT GAACAATTTCCCAA ACAATTTTCATTGG TC

Table 6 summarizes the strains used in the Examples.

TABLE 6 Description of the S. cerevisiae strains used in the examples.Gene Name inactivated Gene overexpressed M2390 (wild-type) None NoneM4652 Δtps1 None M4653 Δtps2 None M11245 None Gene encoding GeneBankAccession XP_748551 (MP244) M10957 None Gene encoding GeneBank AccessionP78617 (MP848) M8841 (described in Δgpd2 Gene encoding SaccharomycopsisWO2011/153516 and Δfdh1 fibuligera glu0111 (GeneBank AccessionWO2012/138942) Δfdh2 CAC83969.1) Δfcy1 Gene encoding the PFLApolypeptide (UnitProtKB Accession A1A239) Gene encoding the PFLBpolypeptide (UnitProtKB Accession A1A240) Gene encoding the ADHEpolypeptide (UnitProtKB Accession A1A067) M11589 Δgpd2 Saccharomycopsisfibuligera glu0111 Δfdh1 (GeneBank Accession CAC83969.1) Δfdh2 Geneencoding the PFLA polypeptide Δfcy1 (UnitProtKB Accession A1A239) Geneencoding the PFLB polypeptide (UnitProtKB Accession A1A240) Geneencoding the ADHE polypeptide (UnitProtKB Accession A1A067) Geneencoding Saccharomyces cerevisiae STL1 (GeneBank Accession NP_010825)M12121 Δgpd2 Gene encoding GeneBank Accession Δfdh1 XP_748551 (MP244)Δfdh2 Gene encoding Saccharomycopsis Δfcy1 fibuligera glu0111 (GeneBankAccession CAC83969.1) Gene encoding the PFLA polypeptide (UnitProtKBAccession A1A239) Gene encoding the PFLB polypeptide(UnitProtKBAccession A1A240) Gene encoding the ADHE polypeptide(UnitProtKB Accession A1A067) Gene encoding Saccharomyces cerevisiaeSTL1 (GeneBank Accession NP_010825) M13913 Δgpd2 Gene encoding GeneBankAccession Δfdh1 XP_748551 (MP244) Δfdh2 Gene encoding SaccharomycopsisΔfcy1 fibuligera glu0111 (GeneBank Accession CAC83969.1) Gene encodingthe PFLA polypeptide (UniProtKB Accession A1A239) Gene encoding the PFLBpolypeptide (UniProtKB Accession A1A240) Gene encoding the ADHEpolypeptide (UniProtKB Accession A1A067)

Fermentation using solid corn mash. Conditions for the results presentedin FIG. 2: the fermentation was performed using 20% total solids (Ts)liquefied corn mash with the addition of 1000 ppm urea, 0.6amyloglucosidase unit (AGU)/grams of total solids (gTs) commercialglucoamylase enzyme, 0.1 g/L dry cell weight (DCW) inoculum, with atotal fermentation time of 51 h. Temperatures were held at 35° C. for 24h and lowered to 32° C. for the remainder of the fermentation. Sampleswere collected and analyzed on HPLC for ethanol titers. Conditions forthe results presented in FIG. 5: the fermentation was performed using25.5% Ts liquefied corn mash with the addition of 500 ppm urea, 0.6AGU/gTs, commercial glucoamylase for M2390, 0.3 AGU/gTs GA for M8841,and 100 μg/ml of purified MP244, 0.3 g/L dry cell weight (DCW) inoculum,with a total fermentation time of 53 h. Temperatures were held at 35° C.for 24 h and lowered to 32° C. for the remainder of the fermentation.Samples were collected and analyzed on HPLC for ethanol titers andresidual trehalose. Conditions for the results presented in FIG. 7: thefermentation was performed using 32% Ts with the addition of 700 ppmurea, 0.48 AGU/gTs commercial glucoamylase for M2390, 0.24 AGU/gTs GAfor M11589 and M12121, 0.3 g/L dry cell weight (DCW) inoculum, with atotal fermentation time of 48 h. The temperature was held at 33° C. for24 h and lowered to 31° C. for the remainder of the fermentation.Samples were collected and analyzed on HPLC for ethanol titers andresidual trehalose.

Extracellular Trehalose Assay. Residual Trehalose was Measure UsingHPLC.

Extracellular trehalase assay. For evaluation of strains expressingsecreted heterologous trehalases, a plate based trehalase assay wasperformed. Strains of interest were 24-72 h in YPD. The cultures werethen centrifuged at 3000 rpm to separate the cells from the culturesupernatant containing the secreted enzymes. The supernatant is thenadded to a 1% trehalose solution in 50 mM sodium acetate buffer (pH5.0). The assay is conducted using a 5:1 trehalose solution:supernatantratio and incubated at 35° C. for 2 h. The reducing sugars were measuredusing the dinitrosalicylic acid reagent solution (DNS) method, using a2:1 DNS:starch assay ratio and boiled at 100° C. for 5 mins. Theabsorbance is measured at 540 nm.

Intracellular trehalose assay. For evaluation of intracellular trehaloseconcentrations, strains were grown in YPD at 35° C. Cells werecentrifuged at 3000 rpm and the supernatant removed, followed by arepeated water wash. Cultures were normalized to the same OD and 0.25 Msodium carbonate added and incubated at 95° C. for 2 h. Next, 0.2 Msodium acetate pH 5.2 was added, followed by the addition of 1 M aceticacid. A total of 0.5 ml of the slurry was treated with 10 μl of ofMegazyme E-trehalase and incubated overnight at 37° C. Glucose wasmeasured using HPLC.

Fermentation using maltodextrin. The fermentation was performed using260 g/L maltodextrin with the addition of 10 g/L yeast extract, 1 g/Lcitrate, 500 ppm urea, 0.6 amyloglucosidase unit (AGU)/grams of totalsolids (gTs) commercial glucoamylase enzyme for the wild type M2390, and0.3 AGU/gTs for the M8841 and M13913 strains, 0.1 g/L dry cell weight(DCW) inoculum, with a total fermentation time of 54 h. Temperatureswere held at 35° C. for 24 h and lowered to 32° C. for the remainder ofthe fermentation. Samples were collected and analyzed on HPLC forethanol titers and residual trehalose.

Example II—Elimination of Key Biosynthetic Genes for the Production ofTrehalose

The material, methods and strains used in this example were presented inExample I.

In order to down regulate/eliminate trehalose production, the nativegenes responsible for the primary synthetic functions (tps1 and tps2)were individually knocked out in the conventional (wild-type) strainM2390. The M4652 and M4653 strains were then evaluated in corn mashfermentation to characterize ethanol production and residual trehalose.As shown in FIG. 2, the Δtps2 strain (M4653) performed well, providingan additional 2.2 g/L of ethanol production over the conventional strain(M2390), coupled with an 86% reduction in residual trehalose (FIG. 3).

Example III—Expression and Secretion of Heterologous TrehalasesTargeting Hydrolysis of Residual Trehalose

The material, methods and strains used in this example were presented inExample I.

In order to target the hydrolysis of residual trehalose in an ethanolfermentation, various heterologous trehalases were cloned by integrating2 copies of the sequence into the conventional yeast host background(M2390) and expressed in S. cerevisiae. The screened heterologoustrehalase sequences are presented in Table 7.

TABLE 7 Amino acid sequence of the heterologous trehalase presented inFIG. 4. Source Accession # SEQ ID NO: MP241 BacillusamyloliquefaciensCCG51384 4 MP242 Debaryomyces hansenii CAG87277 5 MP243 Aspergillusniger CAK43526 6 MP244 Aspergillus fumigatus XP_748551 1 MP245Trichoderma reesei EGR45658 7 MP246 Kluyveromyceslactis CAG99334 8 MP841Schizosaccharomyces pombe NP_595086 9 MP842 Neurospora crassaXP_960845.1 10 MP843 Candida albicans CAA64476.1 11 MP844 Debaryomyceshansenii XP_459109 12 MP845 Candida glabrata AGG12634 13 MP846Kluyveromyces lactis P49381 14 MP847 Rasamsonia emersonii AAQ67343 15MP848 Aspergillus nidulans P78617 2 MP850 Ashbya gossypii NP_984861 16MP851 Magnaporthe oryzae XP_003714173 17 MP853 Thermus thermophilusYP_004082 18

The strains were then screened for secreted trehalase activity using asecreted trehalase assay. Results of the secreted trehalase assay areshown in FIG. 4. The MP244 (from Aspergillus fumigatus, Accession NumberXP_748551 also shown as SEQ ID NO: 1) and MP848 (from Aspergillusnidulans, Accession Number P78617 also shown as SEQ ID NO: 2) trehalasesexhibited increased trehalase activity.

The secreted protein MP244 was His-tagged and purified by FPLC toprovide concentrated volumes of yeast-made isolated enzyme. Afermentation was performed using the M8841 strain, in the presence orthe absence of 100 μg/mL of purified MP244. The results of suchfermentation are shown in FIG. 5. The performance was also compared tothe conventional strain (M2390). The addition of the MP244 trehalaseprovided a 1.25% yield increase over the M8841 strain with no trehalaseadded. The addition of the MP244 trehalase to the M8841 strain provideda total 3.65% yield increase over the conventional strain M2390. Thiswas correlated with the reduction of residual trehalose measured at theend of fermentation (FIG. 6).

Example IV—Expression of Heterologous Trehalase and GlucoamylaseTargeting Ethanol Production

The material, methods and strains used in this example were presented inExample I. The heterologous trehalase gene, MP244 (SEQ ID NO: 1) wasintegrated into the genome of M11589, a strain expressing and secretingthe heterologous glu011 glucoamylase from S. fibuligera and alsopossessing the glycerol reduction pathway The resulting strain, M12121was subjected to a corn mash fermentation and compared to the parentM11589 along with a wild-type strain with no genetic modifications,M2390. As was observed with the exogenous addition of a trehalose, theexpression of the MP244 trehalase provided an additional 1.1% yieldincrease (FIG. 7A) along with a measurable decrease in residualtrehalose (FIG. 7B).

Example V—Maltodextrin Fermentation

Some of the material, methods and strains used in this example werepresented in Example I.

The heterologous trehalase gene, MP244 (SEQ ID NO: 1) was integratedinto the genome of M8841, a strain expressing and secreting theheterologous glu011 glucoamylase from S. fibuligera and also possessingthe glycerol reduction pathway as described in WO 2012/138942. Theresulting strain, M13913 was subjected to a 260 g/L maltodextrinfermentation and compared to the parent M8841 along with a wild-typestrain with no genetic modifications, M2390. As was observed with theexogenous addition of a trehalose, the expression of the MP244 trehalaseprovided an additional 1.42% yield increase over the parent strain,M8841, and a total 3.1% yield increase over the wild type strain (FIG.9A) along with a measurable decrease in residual trehalose (FIG. 9B).While the invention has been described in connection with specificembodiments thereof, it will be understood that the scope of the claimsshould not be limited by the preferred embodiments set forth in theexamples, but should be given the broadest interpretation consistentwith the description as a whole.

REFERENCES

-   WO 2011/153516-   WO 2012/138942-   An M Z, Tang Y Q, Mitsumasu K, Liu Z S, Shigeru M, Kenji K. Enhanced    thermotolerance for ethanol fermentation of Saccharomyces cerevisiae    strain by overexpression of the gene coding for    trehalose-6-phosphate synthase. Biotechnol Lett. 33.7 (2011):    1367-1374.-   Bell W., Sun W., Hohmann S., Wera S., Reinders A., De Virgilio C.,    Wiemken A., Thevelein J M. Composition and Functional Analysis of    the Saccharomyces cerevisiae Trehalose Synthase Complex. Journal of    Bio Chem. 11 (1998): 33311-33319.-   Cao T S, Chi Z., Liu G L., Chi Z M. Expression of TPS1 gene from    Saccharomycopsis fibuligera All in Saccharomyces sp. WO enhances    trehalose accumulation, ethanol tolerance, and ethanol production.    Mol Biotechnol 56.1 (2014): 72-78.-   Elbein A D, Pan Y T, Pastuszak I, Carroll D. New insights on    trehalose: a multifunctional molecule. Glycobiology. 13.4 (2003):    17-27.-   Ge X Y, Xu Y, Chen X. Improve carbon metabolic flux in Saccharomyces    cerevisiae at high temperature by overexpressed TSL1 gene. J Ind    Microbiol Biotechnol. 40 (2013): 345-352.-   Giffen N. New Insights into fermentation drop samples: The real    story of residual sugars. Fuel Ethanol Workshop and Expo.    Minneapolis, Minn. Jun. 5, 2012.-   Guo Z P, Zhang L, Ding Z Y, Shi G Y. Minimization of glycerol    synthesis in industrial ethanol yeast without influencing its    fermentation performance. Metabol Eng 13.1 (2011): 49-59.-   Singer M A and Lindquist S. Thermotolerance in Saccharomyces    cerevisiae: the Yin and Yang of trehalose. Trends Biotechnol. 16.11    (1998): 460-468.-   Thevelain J M. and Hohmann S. Trehalose synthase: guard to the gate    of glycolysis in yeast? Trends Biochem Sci 20.1 (1995): 3-10.

What is claimed is:
 1. A recombinant yeast host cell comprising: (i) afirst genetic modification for reducing the production of glycerol; (ii)a second genetic modification for allowing the production of aheterologous glucoamylase; and (iii) a third genetic modification forallowing the expression of a heterologous trehalase.
 2. The recombinantyeast host cell of claim 1, wherein the first genetic modification isfor reducing the production of one or more native enzymes that functionto produce glycerol.
 3. The recombinant yeast host cell of claim 2,wherein the one or more native enzymes that function to produce glycerolis a glycerol-3-phosphate dehydrogenase 1 (GPD1) polypeptide, apolypeptide encoded by a gpd1 gene ortholog, a glycerol-3-phosphatedehydrogenase 2 (GPD2) polypeptide, a polypeptide encoded by a gpd2 geneortholog, or a combination thereof.
 4. The recombinant yeast host cellof claim 1, wherein the first genetic modification is for reducing theproduction of a FDP1 suppressor (FPS1) polypeptide or a polypeptideencoded by a fps1 gene ortholog, or a combination thereof.
 5. Therecombinant yeast host cell of claim 1, wherein the first geneticmodification is for increasing the production of sugar transporter-like(STL1) polypeptide or a polypeptide encoded by a stl1 gene ortholog, ora combination thereof.
 6. The recombinant yeast host cell of claim 5,wherein the first genetic modification is for expressing the STL1polypeptide, the polypeptide encoded by the stl1 gene ortholog, or acombination thereof from one or more heterologous nucleic acid molecule.7. The recombinant yeast host cell of claim 1, wherein the heterologousglucoamylase is from Rhizopus sp.
 8. The recombinant yeast host cell ofclaim 1, wherein the heterologous trehalase is from Candida sp.,Kluyveromyces sp. or Magnaporthe sp.
 9. The recombinant yeast host cellof claim 7, wherein the heterologous trehalase is from Candida sp.,Kluyveromyces sp. or Magnaporthe sp.
 10. The recombinant yeast host cellof claim 1, wherein the heterologous trehalase has the amino acidsequence of SEQ ID NO: 8 or 13, is a variant of the amino acid sequenceof SEQ ID NO: 8 or 13 having trehalase activity or is a fragment of theamino acid sequence of SEQ ID NO: 8 or 13 having trehalase activity. 11.The recombinant yeast host cell of claim 7, wherein the heterologoustrehalase has the amino acid sequence of SEQ ID NO: 8 or 13, is avariant of the amino acid sequence of SEQ ID NO: 8 or 13 havingtrehalase activity or is a fragment of the amino acid sequence of SEQ IDNO: 8 or 13 having trehalase activity.
 12. The recombinant yeast hostcell of claim 1 being from the genus Saccharomyces sp.
 13. Therecombinant yeast host cell of claim 12 being from the speciesSaccharomyces cerevisiae.
 14. The recombinant yeast host cell of claim 7being from the genus Saccharomyces sp.
 15. The recombinant yeast hostcell of claim 14 being from the species Saccharomyces cerevisiae. 16.The recombinant yeast host cell of claim 10 being from the genusSaccharomyces sp.
 17. The recombinant yeast host cell of claim 16 beingfrom the species Saccharomyces cerevisiae.
 18. A process for convertinga substrate into a fermentation product, the process comprisingcontacting biomass comprising the substrate with the recombinant yeasthost cell of claim 1 under conditions to allow conversion of at least apart of the substrate into the fermentation product.
 19. The process ofclaim 18, wherein the substrate comprises corn, starch, maltodextrin ora combination thereof.
 20. The process of claim 18, wherein thefermentation product is ethanol.